Metabolic Engineering Of Gluconobacter Oxydans 621H On Glucose Utilization And Its Biotransformation Study

Gluconobacter oxydans is widely used in several biotechnological applications such as vitamin C, dihydroxyacetone, and miglitol, because of its abilities of oxidizing many compounds incompletely to accumulate the oxidized products in the culture medium by using membrane-bound dehydrogenases localized on the outer surface of the cytoplasmic membrane. Glucose is not a preferred carbon source for the G. oxydans strain. In the medium with glucose as the sole carbon source, growth of the wild-type strain of G. oxydans is severely inhibited because of the acidic environment caused by oxidation of glucose into gluconic acid. Therefore, high concentration of sorbitol or mannitol is generally used as carbon source. In this study, to solve low pH problem and efficiently use glucose as carbon source for the culture of G. oxydans, an mutant of G. oxydans 621H, deficient in membrane-bound glycose dehydrogenase (mGDH), was constructed (named GDHK) via gene disruption using the cloned gene fragment. The biotransformation property of the mutant strain, as well as the glucose metabolic mechanism of G. oxydans, was also investigated in this study.(1) The knockout vector pSUP202mgdh::Gen was constructed and transferred into G. oxydans by triparental mating. A number of mutant strains were screened to test and verify. When grown in glucose-containing medium, maximum cell density of GDHK (0.783 g DCW/L) was 52%higher than that of the wild-type strain (0.514 g DCW/L), and the Yx/s was improved from 0.049 gDCW/g to 0.141 gDCW/g.(2) Two typical biotransformation systems (glycerol to dihydroxyacetone and glycol to glycolic acid) by different G. oxydans strains grown on glucose, sorbitol, mannitol, and glycerol were studied respectively. The results indicated that the GDHK strain grown on glucose could exhibit similar catalytic performances as the original G. oxydans cultured on other commonly used carbon sources, and was 2-3-folds higher in comparison with the wild-type strain grown on glucose. These results indicate very favorable prospects of using glucose to lower production cost in many important industrial biotransformation processes.(3) Meanwhile, the glucose metabolism of G. oxydans was also partially studied based on the transcriptional information of several related genes. Our results showed that the high transcriptions of the galpz, glkw, sgdh, glnk, g6pdh genes were responsible for glucose utilization of the GDHK strain. These conclusions were confirmed by knocking out three of the above genes and the reconstruction of glucose metabolism of G. oxydans, which provides partially theorical bases for the gorwth enhancement of G. oxydans strains in the medium with glucose as the sole carbons source.